AMP-activated Protein Kinase In Myotubes Research Articles

Potent PDE4 inhibitor activates AMPK and Sirt1 to induce mitochondrial biogenesis.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor. Activation of AMPK leads to a number of metabolic benefits, including improved mitochondrial function in skeletal muscle and lowering of serum glucose levels in type-2 diabetes models. However, direct activation of AMPK leads to cardiac enlargement, and an alternative strategy that activates AMPK without affecting the heart is needed. Inhibition of phosphodiesterase 4 (PDE4), which is poorly expressed in the human heart, activates AMPK in other tissues. In a screen to identify novel PDE4 inhibitors, we discovered compound CBU91, which is 5–10 fold more potent than rolipram, the best characterized PDE4 inhibitor. CBU91, like rolipram, is able to activate AMPK and Sirt1 and increase mitochondrial function in myotubes. These findings suggest that activation of AMPK in myotubes is a general property of PDE4 inhibition and that PDE4 inhibition may activate AMPK in metabolically relevant tissues without affecting the heart.

Iron deficiency attenuates protein synthesis stimulated by branched-chain amino acids and insulin in myotubes

Iron deficiency anemia indicates poor nutrition and is a public health problem. Iron deficiency is also associated with muscle weakness. However, the intracellular mechanisms by which iron deficiency induces muscle weakness are obscure. The purpose of the present study was to evaluate the effect of iron deficiency on protein synthesis in basal and branched-amino acids (BCAA)- and insulin-stimulated state in muscle cells. Differentiated C2C12 myotubes were incubated with an iron chelator, deferoxamine mesylate, and then stimulated with BCAA or insulin to activate protein synthesis. This iron deprivation resulted in a significant reduction in the abundance of iron-containing proteins, such as the mitochondrial complex 1 subunit protein, compared to control cells, but not of protein that does not contain iron, such as citrate synthase. Proteins involved in glucose utilization, such as glucose transpoter-1, hexokinase and AMP-activated protein kinase (AMPK), were upregulated under iron deficiency. Additionally, rates of BCAA- and insulin-stimulated protein synthesis, measured by puromycin incorporation, were lower in iron-deficient myotubes than in control cells. We suggest that low iron availability attenuates BCAA- and insulin-stimulated protein synthesis, possibly via activation of AMPK in myotubes. The present findings advance the understanding of the importance of iron to skeletal muscle protein synthesis and, thus, may contribute to the prevention of sarcopenia and frailty.

Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.

The AMPK activator R419 improves exercise capacity and skeletal muscle insulin sensitivity in obese mice

ObjectiveSkeletal muscle AMP-activated protein kinase (AMPK) is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. R419 is a mitochondrial complex-I inhibitor that has recently been shown to acutely activate AMPK in myotubes. Our main objective was to examine whether R419 treatment improves insulin sensitivity and exercise capacity in obese insulin resistant mice and whether skeletal muscle AMPK was important for mediating potential effects.MethodsGlucose homeostasis, insulin sensitivity, exercise capacity, and electron transport chain content/activity were examined in wildtype (WT) and AMPK β1β2 muscle-specific null (AMPK-MKO) mice fed a high-fat diet (HFD) with or without R419 supplementation.ResultsThere was no change in weight gain, adiposity, glucose tolerance or insulin sensitivity between HFD-fed WT and AMPK-MKO mice. In both HFD-fed WT and AMPK-MKO mice, R419 enhanced insulin tolerance, insulin-stimulated glucose disposal, skeletal muscle 2-deoxyglucose uptake, Akt phosphorylation and glucose transporter 4 (GLUT4) content independently of alterations in body mass. In WT, but not AMPK-MKO mice, R419 improved treadmill running capacity. Treatment with R419 increased muscle electron transport chain content and activity in WT mice; effects which were blunted in AMPK-MKO mice.ConclusionsTreatment of obese mice with R419 improved skeletal muscle insulin sensitivity through a mechanism that is independent of skeletal muscle AMPK. R419 also increases exercise capacity and improves mitochondrial function in obese WT mice; effects that are diminished in the absence of skeletal muscle AMPK. These findings suggest that R419 may be a promising therapy for improving whole-body glucose homeostasis and exercise capacity.

Estradiol and the Estradiol Metabolite, 2‐Hydroxyestradiol, Activate AMP‐activated Protein Kinase in C2C12 Myotubes

Systemic loss of estradiol (E2) during menopause is associated with increased adiposity which can be prevented with E2 replacement. Rodent studies suggest that E2, or lack of, is a key mediator in menopause-related metabolic changes. We have previously demonstrated that E2 treatment produces a rapid, dose-dependent activation of AMP-activated protein kinase (AMPK) in murine skeletal muscle. Activation of AMPK is implicated in the therapeutic benefits of many insulin sensitizing agents including metformin and thiazolidinediones. Here, we expand our observations and provide novel data which demonstrate that in addition to E2, its metabolite 2-hydroxyestradiol (2-HE2), activate AMPK in C2C12 myotubes. C2C12 myotubes were used to examine the effects on E2 and the by-products of its metabolism on AMPK activation. Low concentrations of E2 (10 and 100 nmol/l) were found to increase AMPK phosphorylation by approximately 1.6-fold, while a higher concentration (10 micromol/l) resulted in a approximately 3.0-fold increase. In comparison to E2 treatment alone, incubation of myotubes with E2 and 1-aminobenzotriazole (ABT) (a CYP450 inhibitor that blocks metabolism of E2) caused AMPK activation to be enhanced at low E2 concentrations, but attenuated at higher concentrations. The effects of ABT suggested that one or more E2 metabolites contribute to the maximal activation of AMPK at high E2 concentrations. Indeed, the estrogen metabolite 2-HE2, but not 2-methoxyestradiol (2-ME2), directly activated AMPK in C2C12 myotubes. We propose a model where E2, acting through its metabolite 2-HE2 and the estrogen receptors (ERs), activates AMPK in myotubes. Finally, activation is abolished when all E2 is metabolized to 2-ME2.